The proposed studies are directed at the molecular mechanisms responsible for the regulation of fetal hemoglobin (HbF) and adult hemoglobin (HbA) synthesis in the baboon. Our previous results have demonstrated that a reverse switch from the production of HbA to HbF occurs in baboons subjected to acute erythropoietic stress or treatment with the DNA hypomethylating agent, 5-azacytidine. This reverse switch also occurs in patients with sickle cell anemia and Beta thalassemia who are treated with 5-azacytidine. The clinical effect of increased HbF in these patients was promising, but possible carcinogenicity has hindered further studies. A better understanding of the molecular mechanisms responsible for HbF to HbA switching may indicate better approaches to therapy without producing intolerable side effects. Our studies focus on the role of non-histone nuclear proteins (DNA sequence-specific binding proteins) in controlling HbF expression during gestation. Nuclear proteins will be extracted from nucleated erythroid cells of pre- and post-switch baboon fetuses to determine whether differences in DNA-binding proteins exist in fetuses synthesizing HbF compared to animals synthesizing HbA. DNA binding will be examined using DNA fragments containing known globin gene regulatory sequences. We plan to test for the functional significance of these DNA- binding proteins by in vitro transcription assays, and to purify those proteins that participate in globin gene switching. The regulatory protein genes will be cloned for use in structure and function studies. These cDNA clones will also be used in transfection experiments to evaluate their therapeutic potential.